The overall goal of this Program Project is to develop a multicomponent vaccine for T.cruzi infection and define the optimal means and methods to distribute such a vaccine. During the previous grant period, a heavy emphasis was placed on putting together the infrastructure to test most T.cruzi genes for vaccine efficacy. That infrastructure is now well established and is functioning at high efficacy. The goals of Project 1: Gene Annotation and Cloning, is to keep the machine for gene identification and cloning running, to continue to fine-tune the process and to clone all T.cruzi genes of interest. We will rely on the Bioinformatic core and the T.cruzi Genome Sequencing Consortium (TcGSC) for gene identification and will also use the information derived from the proteomics studies described in Project 2 for identifying and prioritizing genes for cloning and testing. The primary criteria for selection of genes for cloning include: 1) presence in a relatively low number of alleles or variants in the genome, 2) expression in parasites stages that are present in the mammalian host, and 3) presence in the appropriate compartments to serve as effective targets for immune recognition. Genes identified as being as being of interest will be passed on to the Cloning Pipeline in which putative genes are cloned by means of automated primer design, and initial and secondary PCR reactions for the production of adapter sites required for cloning into Gateway entry vectors. Genes are then moved into vaccination vectors for testing in mice. We will clone and test a minimum of 1000 genes per year- and we predict that at this rate we can cover the majority of low copy genes in the T.cruzi genome. Genes will be tested in pools of approximately 96 for the ability to protect mice from lethal infection and protective pools will be further parsed to eventually determine the individual genes that provide the best protection. Genes cloned in this process will be used to produce and test protective cocktails of genes in the vaccination studies described in Project 3.